After creating our biobricks and expressing them in probiotics we needed to test two essential assumptions of our project. The first was that our bacteria can survive a shock of pH and oxygen concentration which they would need to go through when traveling through the digestive system.
The other assumption was that we could create the right growth conditions for the bacteria to produce them in large amounts. The latter point would be necessary in order to mass-produce bacteria commercially which is essential for our entrepreneurial effort
The problem we faced was that we could not control these conditions precisely or without great effort, as a normal incubator allows only control of temperature and shaking frequency. How could we overcome these problems?
Using a bioreactor
We were able to successfully test our assumptions on a larger scale than usually possible in research labs by using a 7l bioreactor. This allowed us to simulate the conditions existing in the transition from one gut-compartment into the other, under controlled conditions.
Secondly, we could show that a large scale production of our probiotic bacteria is possible.
For the fermentation in the bioreactor we used DH5alpha cells transformed with a plasmid containing an expression cassette for our novel biobrick TPH1 downstream of an IPTG promoter.
Before incubating liquid cultures for the fermentation we carried out measurements in LB (Luria Broth) and TB (terrific broth) to establish what medium is ideal for the growth of the bacteria cultures.
We then inoculated the first liquid culture into 250ml shakeflasks using glycerol stocks and gradually scaled up the volume, see picture
We set up the initial parameters for the fermentation, including pH, DO, temperature and rotational frequency. To view the exact set-up of the fermentor, go to Protocols.
Over a period of 15 hours we measured the OD600 of the culture to determine the growth rate. After six hours we induced a pH shock from pH 6.95 to 5 within 45 minutes and afterwards a sudden increase back to base within 10 minutes. Especially the sudden increase was meant to simulate the transition from the stomach into the upper intestine.
Shortly after we induced a DO shock to see whether bacteria could recover from such a shock which is relevant as oxygen concentrations can vary strongly in the lumen of the gut.
Immediately after reducing pH from 6.95 to 5 the culture did not seems to slow down in growth much. The OD measurement following the sudden increase showed a smaller cell density which is likely to be linked to decreased growth.
The OD in the measurement taken an hour after returning the pH of the medium back to 6.96 was higher than immediately after the shock.
The reduction in oxygen concentration was carried out after measuring an increase in OD after the pH induced shock. The cell growth was smaller than during exponential phase after the reduction of oxygen. However, growth still continued at a slower rate after.
The experiment showed that we could successfully grow engineered probiotics at large scale which would allow us to produce engineered bacteria in commercially relevant amounts. The cultures survived the shock of pH, similar to certain transitions in the gut and maintained growing after. Even the complete absence of oxygen flow for half an hour did not kill the culture although diminishing growth.